This invention was made using finds obtained from the U.S. Government and the U.S. Government may therefore have certain rights in the invention.
1. Field of the Invention
The invention generally relates to the fields of cell biology and molecular biology. The invention specifically relates to the detection of apoptosis in biological samples.
2. Related Art
Apoptosis is a process of programmed cell death. Necrosis describes the process of cell death that occurs when cells die for reasons other than apoptosis, such as by severe injury. Apoptosis is characterized by chromatic margination, nuclear condensation and fragmentation, condensation of the cell with preservation of the organelles and plasma membrane integrity, fragmentation of the cell with formation of membrane-bound acidophilic globules (apoptotic bodies), preservation of mitochondrial transmembrane potential and DNA strand breaks (Saraste, 1999, PCT Application No. PCT/US97/21271, Darzynkiewicz et al., 1992). The process of apoptosis has been shown to be regulated by intracellular proteases called caspases (Bantel et al., 2001) The activation of caspases in apoptosis precedes DNA degradation and the development of apoptotic morphology (Saraste, 1999). Necrosis is characterized by depletion of intracellular ATP stores, swelling of the cell with disruption of organelles, rupture of the plasma membrane and loss of mitochondrial transmembrane potential and DNA strand breaks (Saraste, 1999, Darzynkiewicz et al., 1992).
The apoptotic process causes cellular DNA cleavage into multiples of approximately 180 base pairs. This cleavage pattern is caused by the cleavage of the nuclear DNA within the linker regions between nucleosomes. The endonucleases responsible for this cleavage have been shown to have properties similar to DNases I and II (PCT Application No. PCT/US97/21271). Necrosis causes cellular DNA to randomly degrade (PCT Application No. PCT/US97/21271).
Various methods have been used in attempts to detect apoptotic cells. These methods include the terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated dUTP nick end labeling (TUNEL) assay to detect 3′OH groups, measurement of DNA content using DNA-specific fluorophores, assessment of the pattern of DNA fragmentation using gel electrophoresis, evaluation of morphological features such as plasma membrane integrity using probes that are unable to cross the plasma membrane, preservation of mitochondrial transmembrane potential assayed by retention of rhodamine 123, preservation of the ATP-dependent lysosomal proton pump assayed by the supravital uptake of acridine orange (Darzynkiewicz et al., 1992). The previous approaches used to detect DNA breaks could detect free 3′OH groups in cleaved DNA using terminal transferase or label stretches of single-stranded DNA with the help of polymerases.
An in situ ligation approach for selective detection of double-strand (ds) DNA breaks of DNase I-type (bearing 3′OH/5′PO4) in cellular DNA was recently introduced (Didenko and Hornsby, 1996; Didenko et al., 1998; Didenko et al., BioTechniques, 1999). The assay relies on T4 DNA ligase based attachment of double-stranded DNA probes with blunt ends or short 3′ overhangs to the ends of double-strand (ds) DNA breaks that bear 5′PO4. The ligation reaction occurs directly in tissue section (Didenko and Hornsby, 1996; Didenko et al., 1998; Didenko et al., BioTechniques, 1999) or in live cell culture (Didenko et al., Soc. Neurosci., 1999). The probes used in the labeling reaction can be PCR labeled (Didenko and Hornsby, 1996) or synthesized as hairpin-shaped oligonucleotides (Didenko et al., 1998; Didenko et al., BioTechniques, 1999). Because ligase needs a terminal 5′PO4 in the cellular DNA for attachment of the probe, this assay exclusively detects 5′ phosphorylated double strand breaks. An example of a nuclease that can produce such breaks is the key apoptotic nuclease CAD/DFF40 (Widlak et al., 2001).
Several types of DNA breaks are used as markers of apoptosis for detection of apoptotic cells in situ. These breaks in the cellular DNA can possess different terminal configurations. Another type of DNA break is represented by double strand breaks bearing a 5′-OH, which are generated by the ubiquitous DNase II-type lysosomal nucleases (Sikorska and Walker, 1998). DNase II-type nucleases play a role in fundamental biological phenomena such as apoptosis, DNA catabolism, and drug-induced DNA cleavage (Krieser and Eastman, 1998; Barry and Eastman, 1993; Toriglia et al., 1995; Sikorska and Walker, 1998; Bernardi, 1971; Perez-Sala et al., 1995).
However, no ligase can attach the 5′OH end of genomic DNA to the 3′OH end of the probe (Maunders, 1993). Attempting to expand the ligation assay to detection of 5′OH bearing breaks by just adding 5′-phosphates to the probe will not result in labeling of this type of DNA damage due to the probe attachment to 3′OH ends and probe self-ligation.
In the present invention, an approach has been developed for visualization of 5′OH ds DNA breaks directly in tissue sections or in purified cellular DNA in solution, thereby allowing selective detection of a subpopulation of apoptotic cells with DNase II-type fragmentation. The approach also allows the detection of apoptotic cells with DNase I-type and DNase II-type fragmentation simultaneously. The present invention utililizes topoisomerase and combined topoisomerase-ligase-based detection methods to image and analyze specific DNA damage in cells in situ. The importance of selective imaging of individual types of DNA damage is shown by the fact that apoptotic DNase I-type nucleases produce identifiable specific breaks absent in necrotic cells (Didenko and Hornsby, 1996; Didenko et al., 1998). Discrimination of the various types of DNA damage is important in the understanding of the generation and repair of DNA damage, particularly for testing drug toxicity in pharmaceutical production and assessing environmental influences.
Prior to the present invention, there were no methods for the selective detection of a subpopulation of apoptotic cells with DNase II-type fragmentation or for simultaneously detecting DNase I-type fragmentation and DNase II-type fragmentation. The present invention comprises a vaccinia topoisomerase I-based approach to label a double-strand break of DNase II type that bears a 5′OH (Krieser and Eastman, 1998; Barry and Eastman, 1993; Toriglia et al., 1995; Sikorska and Walker, 1998; Bernardi, 1971; Perez-Sala et al., 1995). It also comprises a combination of the ligase and topoisomerase based systems into a single assay, resulting in simultaneous detection of two specific types of DNA damage in situ.